Method for increasing the efficiency of inducing pluripotent stem cells

ABSTRACT

The present invention relates to a method for increasing the efficiency of inducing pluripotent stem cells by utilizing genes Jhdm1b and Jhdm1a that modify histone. By utilizing Jhdm1b, Jhdm1a, and a stem cell inducing factor, the present invention increases the efficiency of inducing pluripotent stem cells and increases the quality of induced pluripotent stem cells. The stem cell inducing factor is a combination of Oct4 and Klf4, or a combination of Sox2, Oct4, and Klf4, or a combination of Oct4 and Sox2, and Oct4 alone. The method further comprises exposing the cells to vitamin C, which further increases the efficiency of inducing pluripotent stem cells as compared with the case where no vitamin C is used. By using less stem cell reducing factors, the method of the present invention reduces the potential carcinogenicity, obtains a high inducing efficiency, and provides high-quality induced pluripotent stem cells capable of germ-line transmission.

SEQUENCE LISTING

A substitute Sequence Listing, incorporated by reference in itsentirety, is provided on identical compact discs (labeled Copy 1Replacement 07/19/2013, Copy 2 Replacement 07/19/2013, andComputer-readable form Replacement 07/19/2013), each compact disccontaining the file 130718_VM41838_PW12048-seq listing-amended-JH.txt,created on Jul. 18, 2013, and is 31.4 KB in size.

BACKGROUND OF THE INVENTION

The present invention relates to a method for increasing the efficiencyof inducing pluripotent stem cells, and more particularly, to a methodfor increasing the efficiency of inducing pluripotent stem cells byutilizing genes Jhdm1b and Jhdm1a that modify histone.

China is a populous country in the world and also has the highest numberof organ losses, damages, failures, and functional disorders as a resultof trauma, disease, aging, and heredity. Classical medical therapiesbased on drugs and surgeries have failed to satisfy the tremendousdemand of clinical medicine. As a result, the research on the stem cellsand the regenerative medicine attracts the attention of numerousresearch entities and all sectors of the society.

The cell transplantation therapy constitutes an important researchdirection of the regenerative medicine, and specific types of celltransplantations may be used to treat heart injury, nervous systemdegenerative diseases, spinal injury, renal failure, hematologicalsystem diseases, and so on. However, the cell transplantation therapy isfacing many intricate problems such as allograft rejection and limitedcell sources.

The stem cell is a type of cell capable of selfrenewal and candifferentiate into various functional cells under certain conditions.Based on their development stage, the stem cells are divided intoembryonic stem cells and adult stem cells. Based on their developmentpotential, the stem cells are divided into three types: totipotent stemcells, pluripotent stem cells, and unipotent stem cells. The stem cellis a type of immature cell that is not fully differentiated and has apotential function to regenerate various tissues and organs and thehuman body, so it is called “universal cell” in the field of medicine.

In order to solve the problems encountered by the cell transplantationtherapy, the transformation of cell fate attracts the attention of moreand more scientists. Although the determination of cell differentiationand fate has always been considered as an irreversible and stableprocess in the development process, there are more and more in vitroevidences showing that this process is reversible.

The study on regulation of cell fate is just in a laboratoryinvestigation state and is far from clinical trial. These transformedcells obtained through over-expression of transcription factors stillhave many application problems, for example, viral sequencesintegration, potential oncogenicity, the purity of the resultanttransdifferentiated cells, and whether they can make up for normal cellswhich are damaged in certain conditions and play their due roles in theorganism.

The induced pluripotent stem cell (iPS) is a type of cell that resembleswith embryonic stem cell and has development totipotency. It acquiresthe properties of the stem cell by inducing the somatic cell throughintroducing specific ES enriched genes. In 2006, Japanese scientistYamanaka introduced 24 candidate genes into mouse fibroblasts by using aretrovirus based vector, screened FBX15 positive cells by means of G418resistance to isolate iPS clones similar to embryonic stem cells, andfinally identified that 4 factors including Oct3/4, Sox2, c-Myc, andKlf4 are sufficient to induce mouse FBX15-iPS cells; as compared withembryonic stem cells, these cells are similar with embryonic stem cellsin the aspects of clone shape, proliferation capability and ability toform teratoma, but they are different from embryonic stem cells in termsof gene expression and genomic methylation profile and cannot obtainliving chimeric mice. Afterwards, this group and other two groupschanged the screening method, and they used Nanog as the standard andobtained iPSs that are similar to embryonic stem cells in many aspectsand these iPSs can produce chimeric offspring. Recently, the threeresearch groups independently confirmed, by tetraploid complementationtest, that mouse iPS cells can develop into an individual and possessdevelopment totipotency.

Following the method of inducing mouse iPSs, in 2007, each of the twogroups Yamanaka [8] and Yu Junying [9] successfully reprogrammed humansomatic cells into iPS cells, wherein the former transduced Oct3/4,Sox2, c-Myc, and Klf4 into human epidermal fibroblasts by using aretrovirus, while the latter incorporated Otc3/4, Sox2, Nanog, and Lin28into foreskin cells by using a lentivirus. Both of the analysis on geneexpression profiling and the analysis on the methylation of the promoterregions of genes Oct3/4 and Nanog showed that the human iPS cell line isvery similar to the corresponding embryonic stem cell line, and all ofthe cells can develop into 3 germinal layers when they are injected intothe body of a nude mouse. Furthermore, somatic cells can be successfullyinduced into iPSs in rat, swine, and monkey, in addition to mouse andhuman.

The cells that can be successfully reprogrammed are not only limited tofibroblasts, and many other types of adult cells can also besuccessfully induced into iPS cells, including pancreas beta cells,adult neural stem cells, hepatocytes, gastric cells, mature B cells,haematopoietic cells, meningocytes, adipose-derived stem cells, cordblood cells, peripheral blood CD34 positive cells, and keratinocytes.For cells at different differentiation stages, the difficulties ininducing and reprogramming them into iPSs are different. Take mousehaematopoietic cells as an example: the reprogramming efficiency ofhaematopoietic stem cells and haematopoietic progenitor cells may be upto 28% which is 300 times that of terminally differentiated T cells andB cells.

In inducing iPSs, it is often to incorporate an exogenous gene intocells by means of a retrovirus and a lentivirus, which provides veryhigh gene transduction efficiency. However, integration of the viralsequence into the genome of the cell may result in gene insertionalmutagenesis and even carcinogenicity, so this gene introduction methodhaving potential risks is obviously unfavorable to application of theiPS technique in the field of regenerative medicine. Therefore, adifferent study group used non-integrating vectors to induce iPSs andsucceeded. These vectors include an adenovirus vector, a commonexpression vector, a transposon, an episome vector, and a minicircle DNAvector.

Both of the combination of Sox2, Klf4, Oct3/4 and c-Myc and thecombination of Sox2, Oct3/4, Nanog and Lin28 can successfully induce thegeneration of iPSs. Further studies found that c-Myc is not essentialfor reprogramming and the three transcription factors including Sox2,Klf4 and Oct3/4 are sufficient to drive the reprogramming of human andmouse somatic cells. Neural stem cells endogenously express high levelsof Sox2, Klf4, and c-Myc, so it only needs to incorporate exogenousOct3/4 in order to successfully induce iPSs. Among the transcriptionfactors used in reprogramming, Sox2, Klf4, and c-Myc can all be replacedby other members of the same family, for example, Klf2 and Klf5 canreplace Klf4; Sox1 and Sox3 can replace Sox2; N-Myc and L-Myc canreplace c-Myc; but Oct1 and Oct6 cannot replace Oct4. Esrrb directlybinds to Oct3/4 protein to regulate the self-regeneration andtotipotency of stem cells, and in reprogramming, Esrrb can replace Klf4to induce iPSs in combination with Sox 2 and Oct3/4. Oct3/4 is a veryimportant transcription factor in reprogramming. Recent studies foundthat nuclear receptors LRH-1 (Nr5a2) and Nr5a1 can replace Oct3/4 andcan induce mouse adult cells into iPSs in combination with Klf4 andSox2.

However, so far, there are several different combinations oftranscription factors capable of reprogramming, including Oct4, Klf4,Sox2, and c-Myc; Oct4, Nanog, Lin28, Sox2; Sox2, Klf4, and Lrh1; Oct4and bmil, as well as reprogramming-related genes such as esrrb and tbx3.For the transcription factor combinations required by existingreprogramming methods, it needs to incorporate as many as 3 or 4transcription factors and the induction efficiency is low. How to reducethe number of transcription factors while maintaining a highreprogramming efficiency is of great importance for reducing theaccumulation of cell mutations in reprogramming and for improving theoperability of the reprogramming technique. Furthermore, searching forgenes that replace common transcription factors facilitates the study ofthe reprogramming mechanism and the improvement of the reprogrammingtechnique.

BRIEF SUMMARY OF THE INVENTION

An objective of the present invention is to provide a method of reducingthe number of transcription factors while maintaining a highreprogramming efficiency, reducing the accumulation of cell mutations inreprogramming, and improving the operability of the reprogrammingtechnique.

To achieve this objective, the following technical solution is adoptedto provide a method for increasing the efficiency of inducingpluripotent stem cells, comprising the following steps:

a. transferring a transcription factor and Jhdm1b into mammalian adultcells which are then cultured in an inducing medium to inducepluripotent stem cell clones, wherein the transcription factor is Oct4alone, or a combination of Oct4, Klf4, and Sox2, or a combination ofOct4, Klf4, c-Myc, and Sox2;

b. culturing and expanding the induced pluripotent stem cell clones in astem cell culture medium.

Another technical solution of the present invention is to provide amethod for increasing the efficiency of inducing pluripotent stem cells,comprising the following steps:

a. transferring a transcription factor and Jhdm1b into mammalian adultcells which are then cultured in an inducing medium containing vitamin Cto induce pluripotent stem cell clones, wherein the transcription factoris Oct4 alone, or a combination of Oct4 and Sox2, or a combination ofOct4 and Klf4, or a combination of Oct4, Klf4, and Sox2, or acombination of Oct4, Klf4, Sox2, and c-Myc;

b. culturing and expanding the induced pluripotent stem cell clones in astem cell culture medium.

Preferably, the above steps are as follows:

a. transferring a transcription factor and Jhdm1b into mammalian adultcells which are then cultured in an inducing medium to inducepluripotent stem cell clones, wherein the transcription factor is Oct4alone, or a combination of Oct4 and Sox2, or a combination of Oct4 andKlf4, or a combination of Oct4, Klf4, and Sox2;

b. culturing and expanding the induced pluripotent stem cell clones in astem cell culture medium.

Preferably, the transcription factor and Jhdm1b are encoded or noncodingRNAs, proteins, or polypeptides capable of inducing pluripotent stemcells.

Preferably, the transferring of Jhdm1b into mammalian adult cells isachieved by incorporating a vector capable of expressing Jhdm1b into thecells.

Preferably, the vector is a viral vector, a plasmid vector, an externalsatellite vector, or an mRNA vector, or is chemically synthesizeddirectly.

Preferably, the viral vector is a retrovirus which is a pMXs vector.

Preferably, the Jhdm1b is a polypeptide for demethylation modification,a functional variant thereof, and a functional fragment thereof.

Preferably, the mammalian adult cells are fibroblasts, neural cells,haematopoietic cells, and neuroglial cells.

Preferably, the mammalian adult cells are mouse embryonic fibroblasts.

Another technical solution is provided by the present invention toprovide a method for increasing the efficiency of inducing pluripotentstem cells, comprising the following steps:

a. transferring a transcription factor, Jhdm1b, and Jhdm1a intomammalian adult cells which are then cultured in an inducing medium toinduce pluripotent stem cell clones, wherein the transcription factor isOct4 alone, or a combination of Oct4 and Sox2, or a combination of Oct4and Klf4, or a combination of Oct4, Klf4, and Sox2;

b. culturing and expanding the induced pluripotent stem cell clones in astem cell culture medium.

Preferably, the above method comprises the following steps:

a. transferring a transcription factor, Jhdm1b, and Jhdm1a intomammalian adult cells which are then cultured in an inducing mediumcontaining vitamin C to induce pluripotent stem cell clones, wherein thetranscription factor is Oct4;

b. culturing and expanding the induced pluripotent stem cell clones in astem cell culture medium containing vitamin C.

The beneficial effects of the present invention are as follows: byutilizing polypeptides Jhdm1b and Jhdm1a that modify histone, and a stemcell inducing factor, the present invention increases the efficiency ofinducing pluripotent stem cells and increases the quality of inducedpluripotent stem cells. The present method achieves better effects byusing less types of stem cell inducing factors as compared with theexisting methods of inducing pluripotent stem cells. Preferably, themethod of the present invention uses Oct4, Klf4 and Sox2, Oct4 and Klf4,Oct4 and Sox2, or Oct4 alone. The method of the present inventionfurther comprises exposing the cells to vitamin C, which furtherincreases the efficiency of inducing pluripotent stem cells as comparedwith the case where no vitamin C is used. By using less stem cellreducing factors, the method of the present invention reduces thepotential carcinogenicity, obtains a high inducing efficiency, andprovides high-quality induced pluripotent stem cells capable ofgerm-line transmission.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows data indicating that Jhdm1a or Jhdm1b increases theefficiency of inducing pluripotent stem cells as mediated by SKO,wherein the control is a pMXs-FLAG empty vector where no gene sequenceis inserted;

FIG. 2 shows data indicating that Jhdm1a or Jhdm1b promotes theefficiency of reprogramming mediated by SKOM;

FIG. 3 shows that in the presence of vitamin C, Jhdm1a and Jhdm1b acttogether to enable re-programming only using SO, KO, and Oct4;

In FIG. 4, a and d are microphotographs of the induced pluripotent stemcells that are finally formed by using Oct4+Jhdm1b (briefed as OB); band e are photographs of chimeric offspring that are developed after theinduced pluripotent stem cells finally formed by using OB are injectedinto the blastula; and c and f are photographs of the offspring that aregenerated after the chimeras mated with wild type mice, wherein thechimeras are developed after the induced pluripotent stem cells finallyformed by using OB are injected into the blastula);

FIG. 5 shows the results of PCR amplification of the genomic DNAs of thepluripotent stem cell clones, indicating that in the genomes of theOB-induced pluripotent stem cell clones C4, C14, C15 and C16, only Oct4and Jhdm1b are integrated, wherein the control is the genomic DNAsextracted from cells infected with Sox2, Klf4, Oct4, c-Myc and Jhdm1b,and MEF indicates genomic DNAs extracted from mouse embryonicfibroblasts;

FIG. 6 shows the results of quantitative PCR, indicating that exogenousgenes of the OB-induced pluripotent stem cell clones C4, C14, C15 andC16 are silently expressed, wherein the OB D4 control is a cDNA templateobtained by reverse transcription of mRNAs extracted from the cells thathave been infected with Oct4 and Jhdm1b and cultured for 4 days, and MEFis the mouse embryonic fibroblast;

FIG. 7 shows the results of real-time quantitative PCR, indicating thatthe OB-induced pluripotent stem cell clones C4, C14, C15 and C16 expressembryonic stem cell specific genes, wherein R1 is the mouse embryonicstem cell line, and MEF is the mouse embryonic fibroblast;

FIG. 8 shows the results of immunofluorescence, indicating that theOB-induced pluripotent stem cell clone C14 expresses embryonic stem cellspecific gene Rex1 and embryonic stem cell specific surface markerSSEA-1, wherein Marker represents a stem cell specific marker molecule(i.e., Rex1 or SSEA-1);

FIG. 9 shows the analysis results of measured methylation of CpGs in aregion of Oct4 adjacent to the promoter in mouse embryonic fibroblastsand induced pluripotent stem cells;

FIG. 10 shows the analysis results of measured methylation of CpGs in aregion of Nanog adjacent to the promoter in mouse embryonic fibroblastsand induced pluripotent stem cells;

FIG. 11 shows the karyograms of Oct4 and Jhdm1b induced pluripotent stemcells;

FIG. 12 shows the efficiencies of inducing pluripotent stem cells by thevarious mutants of Jhdm1b. The Jmjc mutation involves mutating histidineat position 221, isoleucine at position 222, and aspartic acid atposition 223 into alanine; the CxxC mutation involves mutating cysteineat positions 586, 589, and 592 into alanine;

FIG. 13 shows the spectrum of the pMXs-FLAG plasmid.

DETAILED DESCRIPTION OF THE INVENTION

All the technical terms used herein have the same meanings as understoodby those of ordinary skills in the art. For the definitions and terms ofthe art, one killed may refer to, for example, Current Protocols inMolecular Biology, edited by Ausubel, et al, John Wiley & Sons, 2009.The abbreviations of amino acid residues are the standard 3-lettersand/or 1-letter codes that are used in the art to represent one of the20 common L-amino acids.

In spite of the numerical ranges and parameter approximations shown inthe broad scope of the present invention, the values shown in thespecific embodiments shall be recorded as accurate as possible. However,any values must contain certain error by themselves inevitably, whichare attributable to their standard deviations present in theirrespective measurements. In addition, all the ranges disclosed hereinshall be construed as covering any and all sub-ranges thereof.

The terms “polypeptide” and “protein” used herein may be usedinterchangeably to indicate a string of at least two amino acid residuesthat are interconnected with one another via covalent bond (e.g.,peptide bond), which may be recombinant polypeptides, naturalpolypeptides, or synthetic polypeptides. Particularly, the polypeptidesdescribed herein are human and/or mouse polypeptides.

The terms “variant”, “polypeptide variant” or “analogue” used hereinindicates a polypeptide that is different from the original polypeptidein the amino acid sequence by one or more substitutions, deletions,insertions, fusions, truncations or any combinations thereof. Thevariant polypeptide may be fully functional or may lack one or moreactive functions. The term “functional variant” used herein onlycontains, for example, conservative changes or the changes innon-critical residues or non-critical regions, and retains the functionsof the original polypeptide. The functional variant may further containthe substitution of similar amino acids, which results in unchangedfunctions or insignificant function changes. Amino acids that areimportant for the functions may be identified by methods known in theart, for example, site directed mutagenesis or glycine scanningmutagenesis (Cunningham, B. and Wells, J., Science, 244: 1081-1085,1989). Sites that are crucial to polypeptide activity may be determinedby, for example, structural analysis such as crystallization, nuclearmagnetic resonance, or photoaffinity labeling (Smith, L. et al., J. Mol.Biol., 224: 899-904, 1992; de Vos, A. et al., Science, 255: 306-312,1992).

In some embodiments of the present invention, the variants of Jhdm1a areselected from polypeptides comprising an amino acid sequence that is atleast 70% (preferably 80%, 90%, 95%, 98%, and 99%) homologous to theamino acid sequence encoded by SEQ ID NO: 1. In other embodiments of thepresent invention, the variants of Jhdm1b are selected from polypeptidescomprising an amino acid sequence that is at least 70% (preferably 80%,90%, 95%, 98%, and 99%) homologous to the amino acid sequence encoded bySEQ ID NO: 2. The amino acid sequence encoded by Jhdm1a is SEQ ID NO: 7,and the amino acid sequence encoded by Jhdm1b is SEQ ID NO: 8.

The term “fragment” used herein refers to a molecule that is only a partof a full-length sequence. For example, a Jhdm1b polypeptide fragment istruncated Jhdm1b. The fragments may contain a sequence from any end ofthe full-length sequence or a sequence from the middle of thefull-length sequence. The fragment may be a “functional fragment”, forexample, a fragment that retains one or more functions of thefull-length polypeptide. The term “functional fragment” used hereinindicates that said fragment retains the functions of the full-lengthpolypeptide, for example, inducing pluripotent stem cells or increasingthe efficiency of inducing pluripotent stem cells.

Unless otherwise stated, when polypeptides, nucleic acids, or othermolecules are mentioned herein, they include functional variants andfunctional fragments. For example, Jhdm1b and Jhdm1a further indicatethe functional variants and functional fragments of natural Jhdm1b andJhdm1a respectively.

The term “Jhdm1b” used herein may indicate a member of the family ofJmjC-domain-containing histone demethylase (JHDM) that is evolutionarilyconserved and widely expressed. It is also called Fbx110. In particular,said polypeptide is a human and/or mouse polypeptide.

The term “Jhdm1a” used herein may indicate another member of the familyof JmjC-domain-containing histone demethylase (JHDM). It is also calledFbx111. In particular, said polypeptide is a human and/or mousepolypeptide.

The term “induced pluripotent stem cells” or “iPSs” used herein may beused interchangeably to indicate pluripotent stem cells obtained byartificially inducing non-pluripotent cells (such as somatic cells).Said inducing is generally achieved by forced expression of a specificgene, and this process is also called “inducing cells into pluripotentstem cells” herein.

The term “stem cell inducing factor” used herein indicates a factor thatis capable of inducing cells into pluripotent stem cells by itself aloneor in combination with other factors, such as proteins, polypeptides,and encoded or noncoding RNAs. Preferably, the stem cell inducing factoris a transcription factor, including Oct-3/4, the members of Sox family,the members of Klf family, the members of Myc family, Nanog, LIN28 andthe like. Preferably, the stem cell inducing factor is selected from oneor more of Oct4, Klf4, Sox2, and c-myc. More preferably, the stem cellinducing factor includes at least Oct4. In particular, the polypeptideis a human and/or mouse polypeptide.

The term “Oct4” used herein indicates a member of the family of octamertranscription factors. It plays a crucial role in maintaining thepluripotency of the cells. In the literatures, Oct4 was also calledOct3.

The term “Klf4” used herein indicates a member of the KrOppel-likefamily of transcription factors.

The term “Sox2” used herein indicates a member of the family of Soxtranscription factors.

The term “c-myc” used herein indicates a transcription factor that iswell known by those skilled in the art. It regulates the expression ofmany genes and recruits histone transacetylase. Its mutations arerelated to many cancers.

The term “histone modification” used herein indicates a variety ofmodifications to histone, such as acetylation, methylation,demethylation, phosphorylation, adenylation, ubiquitination, and ADPribosylation. In particular, the histone modification includes thedemethylation of histone.

The term “object” used herein refers to mammals, such as human being.Other animals may also be included, for example domestic animals (e.g.,dog and cat), poultry (such as cattle, sheep, swine, and horse), orlaboratory animals (such as monkey, rat, mouse, rabbit, and guinea pig).

The term “consistency”, “percent consistency”, “homology”, or “identity”used herein refers to the sequence identity between two amino acidsequences or nucleic acid sequences. The percent consistency may bedetermined by the alignment of two sequences, and it refers to thenumber of identical residues (i.e., amino acids or nucleotides) atpositions common to the compared sequences. Sequence alignment andcomparison may be carried out by the standard algorithms of the art (forexample, Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman andWunsch, 1970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc.Natl. Acad. Sci., USA, 85:2444) or a computerized version of thesealgorithms (Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 575 Science Drive, Madison, Wis.). The computerizedversion is publicly available as BLAST and FASTA. Additionally, theENTREZ available from the National Institute of Health (Bethesda Md.)may be used for sequence comparison. When BLAST and gapped BLASTprograms are used, the default parameters of the respective programs(such as BLASTN, which is available on the internet site of the NationalCenter for Biotechnology Information) may be used. In one embodiment,GCG with a gap weight of 1 may be used to determine the percent identitybetween two sequences, such that each amino acid gap is given a weightas if it is a single amino acid mismatch between the two sequences.Alternatively, ALIGN program (version 2.0), which is a part of GCG(Accelrys, San Diego, Calif.) sequence alignment software package, maybe used.

The term “vector” used herein is used in the meaning well known by thoseskilled in the art and may be an expression vector. The vector mayinclude viruses (such as poxvirus, adenovirus, and baculovirus); yeastvectors, bacteriophages, chromosomes, artificial chromosomes, plasmids,cosmids, episome vectors, and mRNA vectors, or may be chemicallysynthesized directly. Preferably, the virus vector is a retrovirusand/or lentivirus vector. More preferably, the retrovirus is a pMXsvector.

The term “excessive” used herein indicates being significantly higherthan the normal level, and particularly indicates that the expression ofa polypeptide is statistically significantly higher that in normalcells. Preferably, it is higher by 20%, 50%, 100%, 200%, or even 5, 10,or 100 times.

The term “over-expression” used herein indicates that the expressionlevel is significantly higher than the normal level, and particularlyindicates that the expression of a polypeptide is statisticallysignificantly higher that in normal cells. Preferably, it is higher by20%, 50%, 100%, 200%, or even 5, 10, or 100 times.

The term “incorporation” used herein indicates a process to introduceexogenous substances (such as nucleic acids or proteins) into cells by,for example, calcium phosphate transfection, virus infection, liposometransfection, electroporation, gene gun or the like.

Herein, delivering an exogenous polypeptide into cells may be carriedout by various methods, for example, by transporters or transportfactors, and preferably, by liposome, bacterial polypeptide fragments orthe like (refer to WO2002/079417, the content of which is incorporatedherein by reference).

The cells that may be used in the method of the present invention arepreferably mammalian cells, and more preferably human and mouse cells.In particular, the cells are somatic cells, such as epithelial cells,neural cells, fibroblasts, endothelial cells, myocytes, haematopoieticcells, immunocytes, and lymphocytes. More particularly, the cells arepancreatic beta cells, adult neural stem cells, hepatocytes, gastriccells, mature B cells, haematopoietic cells, meningocytes,adipose-derived stem cells, cord blood cells, peripheral blood CD34positive cells, and keratinocytes.

Example 1 1. Construction of Vectors Comprising Jhdm1a and Jhdm1b CodingRegions

a. Design of Primers for Cloning

The sequence data of cDNAs of Jhdm1a and Jhdm1b were obtained fromhttp://www.ncbi.nlm.nih.gov/pubmed, wherein the sequence of the cloningregion of the Jhdm1a cDNA is SEQ ID NO: 1, and the sequence of thecloning region of the Jhdm1b cDNA is SEQ ID NO: 2. The coding sequencesof Jhdm1a and Jhdm1b were amplified by designing specific primers.

The base sequence of Jhdm1a upstream primer is shown as SEQ ID NO: 3;

The base sequence of Jhdm1a downstream primer is shown as SEQ ID NO: 4;

The base sequence of Jhdm1b upstream primer is shown as SEQ ID NO: 5;

The base sequence of Jhdm1b downstream primer is shown as SEQ ID NO: 6;

b. Amplification of Coding Sequences by RT-PCR

Total mRNAs were extracted from isolated ICR mouse embryonic fibroblasts(MEFs) and human H1 embryonic stem cells according to the followingmethod: a culture medium was removed from a culture tray, cells wererinsed with 3-5 ml of normal saline (PBS) (Gibco), and the rinsingsolution is discarded. Then, 1 ml of cell lysate Trizol (Takara) wasadded into the culture tray, and a pipet was used to draw the resultingmixture solution and gently blow the cells so that they were completelydissolved in the lysate. Next, they were transferred into a clear 1.5 mlcentrifuge tube to be stored at −80° C. or immediately subjected to thefollowing extraction step. Afterwards, 200 μl of trichloromethane wasadded thereto, and the mixture were well mixed by turning the tube upand down for about 30 seconds and then centrifuged at 4° C. at 12000 rpmfor 5 minutes. The supernatant was carefully drawn and transferred intoa clear 1.5 ml centrifuge tube and then an equal volume of isopropanolwas added thereto. They were well mixed, allowed to stand at the roomtemperature for 5 minutes, and then centrifuged at 4° C. at 12000 rpmfor 5 minutes, at which time a small white precipitate was found at thebottom of the tube. The supernatant was discarded carefully, and next,500 μl of an 80% ethanol solution was added into the tube to rinse awayresidual isopropanol. The tube was centrifuged at 12000 rpm to removethe ethanol solution. The centrifugate was kept at the room temperaturefor 30 minutes so that the white total mRNAs at the bottom of the tubewere fully dried. Next, 30-50 μl of double distilled water was addedinto the centrifuge tube to incubate at 55° C. for 30 minutes.Afterward, the tube was taken out and measured for the concentration oftotal mRNAs by a spectrophotometer. The extracted total mRNAs werestored at −80° C. or directly used for preparing cDNAs by reversetranscription for later use.

The specific process and method of reverse transcription are as follows.Generally, 1 μg of total mRNAs were taken for reverse transcription, towhich oligodT (Takara), dNTP (Takara), RTace (Toyobo), RT buffer and RRI(RNAse inhibitor, Takara), and RNase/DNase-free water were added. Themixture reacted on a PCR instrument at 42° C. for 60 minutes, wasincubated at 98° C. for 5 minutes, and was then cooled down to the roomtemperature. After the reverse transcription succeeded, 0.5 μl of thereaction was taken out to act as the template to amplify the target geneby PCR using the primers designed by the above method. The reagents usedinclude high-fidelity polymerase KOD and its buffer (Toyobo), dNTPs(Takara), and primers. The following process was run on the PCRinstrument: denaturation at 96° C. for 5 minutes, at 95° C. for 30seconds, annealing at 60° C. for 25 seconds, and elongation at 68° C.for 3.5 minutes; the 2-4 steps were repeated for 32 times.

c. Plasmid Construction

Please refer to FIG. 13. After the amplification was completed, the PCRproduct was subjected to agarose gel electrophoresis and PCR fragmentswere extracted using a gel extraction kit (TIANGEN, DP214-03). The pMXsvector (purchased from addgene, and inserted with multiple cloning sitesand FLAG labeling sequences) was used. The modified pMXs vector iscalled pMXs-FLAG, the plasmid presentation of which is shown in FIG. 13.The vector was cleaved with pmel and dephosphorylated with calfintestinal alkaline phosphatase (CTAP) so as to avoid its self-ligation.The treated vector was recovered using the gel extraction kit (TIANGEN,DP214-03) for later use. The pMX-FLAG vector and the gene fragments ofJhdm1a/Jhdm1b were ligated by a ligation kit (Takara, DNA Ligation Kit),and then the ligating product was used to transform competent E. Coli.The positive clones were selected, the plasmids were extracted andsequenced, and finally plasmids were prepared in a large scale.

2. Introducing the Coding Sequences of Jhdm1a/Jhdm1b and the PluripotentStem Cell Inducing Factor (Transcription Factor) into Mouse EmbryonicFibroblasts

Unless specifically stated, the mouse based somatic cell reprogrammingwas carried out by the following manner in all cases.

Culture Medium

The culture medium for feeder layer cells, MEF cells, and PlatE cellsconsists of: high glucose basal medium DMEM (Gibco), plus 10% fetalbovine serum (FBS, PAA).

Inducing medium: the present invention used an inducing medium that isconventional in the laboratory, and the composition of a preferredinducing medium includes DMEM high glucose medium (Gibco), 15% fetalbovine serum (FBS, Gibco), 0.1 mM nonessential amino acid (NEAA, Gibco),2 mML-glutamine (Glutamax, Gibco), 1 mM sodium pyruvate (Gibco), 55 μMβ-mercaptoethanol (β-ME, Gibco), penicillin (50 U/mL) and streptomycin(50 μg/mL), leukemia inhibitory factor 1000 U/ml (LIF, Millipore), andas necessary, 50 μg/mL vitamin C (sigma).

Stem cell culture medium: the present invention used a stem cell culturemedium that is conventional in the laboratory, and preferably the mESstem cell culture medium, the composition of which consists of: highglucose DMEM medium (Gibco) supplemented with 15% fetal bovine serum,0.1 mM nonessential amino acid (NEAA, Gibco), 2 mM L-glutamine(Glutamax, Gibco), 1 mM sodium pyruvate (Gibco), 55 μM β-mercaptoethanol(Gibco), penicillin (50 U/mL) and streptomycin (50 μg/mL), and leukemiainhibitory factor 1000 U/ml (LIF, Millipore). 50 μg/mL vitamin C (sigma)are incorporated as necessary.

KSR serum-free culture medium: KSR, the abbreviation of Knockout SerumReplace, is a commercialized serum replacing stem cell culture additiveand is used as a complete KSR serum-free medium for culturing stem cellsor iPS clones, the composition of which consists of: KNOCKOUT DMED (abasal medium with optimized osmotic pressure that is suitable forculturing stem cells), a 15% KSR additive, 0.1 mM nonessential aminoacid (NEAA, Gibco), 2 mM L-glutamine (Glutamax, Gibco), 1 mM sodiumpyruvate (Gibco), 55 μM β-mercaptoethanol (β-ME, Gibco), penicillin (50U/mL) and streptomycin (50 μg/mL), leukemia inhibitory factor 1000 U/ml(LIF, Millipore). All the iPS processes and cloning culture media aresupplemented with mouse leukemia inhibitory factor (LIF, millipore,trade name: ESGRO, a growth factor that inhibits the differentiation ofmouse stem cells) at a final concentration of 1000 U/ml.

3. Cells for Reprogramming

All the somatic cells for reprogramming are OG2 mouse embryonicfibroblasts (homemade), the passage number of which does not exceedthree. One property of the OG2 mouse is that there is a greenfluorescence protein (GFP) under the control of the Oct4 promoter thatis specifically expressed by the stem cells. In reprogramming, when theendogenous Oct4 of the OG2 mouse embryonic fibroblasts is activated, thegreen fluorescence protein is expressed concomitantly. As observedthrough a fluorescence microscope, the successfully reprogrammed cellsor cloned cell aggregates are green, and it is easy to compare thereprogramming efficiencies at different conditions by directly adding upthe number of reprogrammed clones, i.e., the number of greenfluorescence clones, or by analyzing the proportion of greenfluorescence cells through a flow cytometer.

The reprogrammed cells were prepared as follows. The cells were seededin 12-well plates (Corning) at a density of 20000 cells per well, andafter 6-18 hours, were infected with viruses with the mousereprogramming factor based on the density and the state of the cells.

4. Preparation of Viruses

The transcription factors for reprogramming include the retrovirusvector pMXs for cDNAs of mouse Oct4, Sox2, Klf4, and c-Myc (fromAddgene, numbered Plasmid 13366, Plasmid 13367, Plasmid 13370, andPlasmid 13375 respectively); Oct4, NCBI accession number: NM_(—)013663;Sox2, NCBI accession number: NM_(—)011443; Klf4, NCBI accession number:010637; and c-Myc, NCBI accession number: NM_(—)001177353. Thereprogramming factor plasmids on the pMX vector were transfected intothe viral packaging cells (PlatE) by using a homemade calcium phosphatetransfection reagent, and the specific process is: 7500000 PlatE cellswere seeded in a 10-cm-diameter culture tray (Corning), and 12 hourslater, the old culture medium was replaced by 7.5 ml of a culture mediumfree of penicillin/streptomycin, and then the cells were placed into anincubator. Next, the transfection mixture was prepared: 25 μg of theplasmids were taken and placed into a 15 ml centrifuge tube, and thereto156.25 μl of a 2 M calcium chloride solution was added sequentially andan appropriate amount of water was additionally added so that the totalvolume thereof was 1.25 ml; they were well mixed, and 1.25 ml of an HBSsolution was added thereto; the resulting mixture was mixed wellimmediately, allowed to standstill for 2 minutes, and then addeddropwise into a PlatE culture tray and well mixed. 9-12 hours after thetransfection, the old culture medium was replaced by 10 ml of a freshculture solution; 48 hours after the transfection, the culture solutionwas collected and filtered with a 0.45 μm filter membrane to be used asthe viral solution for first infection; thereto a fresh culture solutionwas added 24 hours later, and the culture solution was re-collected inthis way to be used as the viral solution for second infection.

5. Infecting MEF Cells with the Virus

The infection was carried out in two rounds, wherein the inducingfactors used infected the cells simultaneously, each well of the 12-wellplates was infected with 1 ml of the virus, the second round ofinfection was carried out 24 hours after the first round of infection,and the viral solution was replaced with the mES culture medium (asdescribed above) 24 hours after the second round of infection. The dayof solution replacement was recorded as day 0 (D0); at different timepoints after infection, in the original wells, the number of GFPfluorescence clones was counted or the proportion of GFP fluorescencecells was analyzed using a flow cytometer as required by the experiment.

6. Culturing the Infected Cells Until Formation of Stem Cell Clones

Embryonic stem cell-like monoclones with a swollen shape and clear edgeswere picked out using glass needles and directly transferred intoculture plates (Corning) laid with feeder layer cells (the feeder layercells are ICR mouse fibroblasts treated with mitamycin) in advance toculture with the KSR culture medium. At day 2 after infection, theculture system was replaced with a fresh inducing medium, and afterward,the inducing medium was replaced everyday until the experiment wascompleted.

Based on the above method for producing stem cell clones, theexperiments were carried out using different combinations of pluripotentstem cell inducing factors.

The combinations of pluripotent stem cell inducing factors are describedas follows:

The combination of Klf4, Sox2, c-Myc, and Oct4 is abbreviated as SKOM.

The combination of Klf4, Sox2, and Oct4 is abbreviated as SKO.

The combination of Klf4 and Oct4 is abbreviated as KO.

The combination of Sox2 and Oct4 is abbreviated as SO.

The combination of Oct4 and Jhdm1b is abbreviated as OB.

C4, C14, C15, and C16 are the four clones picked out from the OB inducedreprogrammed cells.

The results of the experiments using the combinations of stem cellinducing factors are as follows:

Referring to FIG. 1, no matter whether vitamin C was present or not,Jhdm1a or Jhdm1b obviously improved the efficiency of reprogramming, andin the presence of vitamin C, the improvement was more significant. FIG.1 shows the data that Jhdm1a or Jhdm1b improved the efficiency ofinducing pluripotent stem cells as mediated by SKO, wherein the controlis a pMXs-FLAG empty vector with no gene sequence inserted therein;

Referring to FIG. 2, no matter whether vitamin C was present or not,Jhdm1a or Jhdm1b obviously improved the efficiency of reprogramming, andin the presence of vitamin C, the improvement was more significant. FIG.2 shows the data that Jhdm1a or Jhdm1b improved the efficiency ofinducing pluripotent stem cells as mediated by SKOM, wherein the controlis a pMXs-FLAG empty vector with no gene sequence inserted therein;

Referring to FIG. 3, in the presence of vitamin C, Jhdm1a and Jhdm1bacted together to enable the induction of pluripotent stem cells only inthe availability of SO, KO, or Oct4, wherein mESC+Vc indicates that theculture medium used in the induction is stem cell culture medium mESsupplemented with 50 μg/ml vitamin C, and the control is a pMXs-FLAGempty vector.

Therefore, the present invention reaches a conclusion that Jhdm1a andJhdm1b can significantly improve the efficiency of inducing pluripotentstem cells, greatly reduce the types of transcription factors requiredto be incorporated while maintaining a high reprogramming efficiency,which provides great benefits for reducing the accumulation ofreprogrammed cell mutations and reducing their carcinogenecity. Inaddition, the method of the present invention also improves theoperability of the reprogramming technique, reduces the operativedifficulty, and facilitates subsequent medical applications.

Example 2 Identification of the Induced Pluripotent Stem Cells from theExample 1

As shown in FIG. 3 and FIG. 6 to FIG. 10, a series of identificationexperiments were carried out on pluripotent stem cell clones induced byOct4 and Jhdm1b to verify whether they are iPS cells (inducedpluripotent stem cells). The identification experiments include:quantitative PCR, immunofluorescence assay of their surface markers,promoter methylation degree analysis, karyotype identification, chimeraformation, and so on.

Quantitative PCR Experiments:

All the quantitative PCR experiments were conducted in a CFX-96 typequantitative PCR instrument from Biorad using a kit from Takara, and thereaction conditions were 95° C., 2 minutes, 95° C., 10 seconds, 60° C.,30 seconds, reading the fluorescence value, repeating for 40 cycles.

Analysis of Methylation of the Promoter Region

The analysis was carried out by sodium bisulphite sequencing. Thegenomic DNAs in the target cells were extracted (Promega, Wizard®Genomic DNA Purification Kit), the concentration was measured,approximately 2 μg of DNAs were placed into a 1.5 ml EP tube where theywere diluted with ddH20 to 50 μl, thereto 5.5 μl of freshly prepared 3 MNaOH was added, and the resulting solution was treated in a water bathat 42° C. for 30 minutes; next, the solution was taken out, 30 μl of 10mM hydroquinone (sigma) was added into the mixture solution after thewater bath treatment, and then 520 μl of 3.6 M sodium bisulphite (Sigma,S9000) was additionally added into the solution after the water bathtreatment; the EP tube was wrapped with aluminum foil paper outside toavoid light, and the solution was well mixed by gently turning the EPtube up and down; 200 μl of paraffin oil was added thereto so as toprevent evaporation of water and oxidization, and the solution wastreated in a water bath at 50° C. in darkness for 16 hours.

Next, the tip of a pipet was put below the paraffin oil layer to drawthe mixture solution into a clear 1.5 ml centrifuge tube, and themodified DNAs were recovered using a Promega Wizard Cleanup DNApurification and retraction system (Promega, A7280), and then stored at−20° C. or subjected to a further experiment. 50 ng of the aboveextracted DNAs were taken as the template to conduct the PCR reaction.Afterward, the PCR product was recovered by gel extraction (TIANGEN,DP214-03), and the PCR product and a T vector (Takara) were then ligatedand transformed. The positive clones were selected and sent to asequencing company for sequencing, and the results were compared tostatistically analyze the methylation of the CpG islands.

Identification of Karyotype of iPS Cells

The identification of karyotype of iPS cells was conducted according tothe following method: 2-3 hours before harvesting, to the cells to beanalyzed for karyotype, 0.1 ml of 5 μg/ml colchicine (commerciallyavailable, with a final concentration of 0.1 μg/ml) was added. They werewell mixed, further cultured for 2-3 hours, transferred into a 10 mlcentrifuge tube, and then centrifuged at 1500-2000 rpm for 10 minutes.The supernatant was discarded, and an 8 ml of a hypotonic solution(0.075 M KCl, preheated at 37° C.) was added to the tube. The cellprecipitate was blown homogenously and placed in an incubator at 37° C.for half an hour. Thereto, 1 ml of a freshly prepared stationary liquid(a mixture of methanol and glacial acetic acid at a volume ratio of 3:1,commercially available) was added. The resulting mixture was gentlymixed and centrifuged at the same rotation speed for the same period oftime as the above. The supernatant was drawn off. 8 mL of the stationaryliquid was added thereto. The cells were adequately mixed, fixed at theroom temperature for at least half an hour, and centrifuged again. Thesupernatant was discarded, and a fresh stationary liquid was addedthereto to further fix the cells for at least half an hour (desirablyovernight). To the cell precipitate obtained after centrifugation andremoval of the supernatant, about 0.2 ml of a fresh stationary liquidwas added, and the resulting mixture was well mixed. The resulting cellsuspension was dropped onto pre-cooled slides (it is advisable to drop 3drops of the cell suspension on each slide) which were then baked on analcohol lamp. The cells were then cooled down and banded.

Blastula Chimera Test

In the blastula chimera test, iPS cells were injected into theblastocoele of donor mice, and then the injected blastulas weretransplanted into the uteruses of pseudopregnant female mice to makechimeric mice. Whether the born mice produce chimera was determinedbased on their coat color.

Experiments were carried out according to the above method, and theresults are analyzed as follows:

Referring to FIG. 5, the PCR amplification results of the genomic DNAsof pluripotent stem cell clones show that, in the genomes of OB-inducedpluripotent stem cell clones C4, C14, C15, and C16, only Oct4 isintegrated with Jhdm1b, wherein the control is genomic DNAs extractedfrom cells infected with Sox2, klf4, oct4, cMyc, and Jhdm1b, and MEFindicates genomic DNAs extracted from mouse embryonic fibroblasts;

Referring to FIG. 6, the results of quantitative PCR show that exogenousgenes of the OB-induced pluripotent stem cell clones C4, C14, C15, andC16 were silently expressed, wherein the OB D4 control is a cDNAtemplate obtained by reverse transcription of mRNAs extracted from cellsthat have been infected with Oct4 and Jhdm1b and cultured for 4 days,and MEF is mouse embryonic fibroblast;

Refer to FIG. 7. As shown in FIG. 7, the results of real-timequantitative PCR show that the OB-induced pluripotent stem cell clonesC4, C14, C15, and C16 expressed embryonic stem cell specific genes,wherein R1 is mouse embryonic stem cell line, and MEF is mouse embryonicfibroblast; the expression level of endogenous embryonic stem celltranscription factors in the stem cells obtained by using thecombination of Oct4 with Jhdm1b was substantially consistent with thatin the embryonic stem cells. This indicates that the OB-inducedpluripotent stem cell clones C4, C14, C15, and C16 express embryonicstem cell specific genes, and thus indicates that the pluripotent stemcells induced by the method of the present invention have thecharacteristics of pluripotent stem cells.

Refer to FIG. 8. As shown in FIG. 8, the immunofluorescence results showthat the pluripotent stem cells obtained through OB expressed SSEA-1 onthe surface and expressed Rex1 as well.

Refer to FIG. 9 which shows the analysis of methylation of CpG islandsin the Oct4 promoter region, wherein the CpG islands of the donor cellswere methylated, while the CpG islands at the corresponding positions ofthe induced pluripotent stem cells were significantly demethylated.

Refer to FIG. 10 which shows the analysis of methylation of CpG islandsin the Nanog promoter region, wherein OB-C14, OB-C15, and OB-C16 arethree pluripotent stem cells induced by Oct4 and Jhdm1b. The black partsindicate methylation, and the white parts indicate absence ofmethylation. The CpG islands of the donor cells were methylated, whilethe CpG islands at the corresponding positions of the inducedpluripotent stem cells were significantly demethylated; Nanog and Oct4are genes that are specifically expressed by embryonic stem cells andtheir expression states are closely related to the cell fate. Theseresults show that the cells obtained by using the OB group had changedfate, that is to say, they were induced into pluripotent stem cells.

Refer to FIG. 11 which shows that the stem cells obtained by the methodof the present invention had normal karyotypes, wherein OB-14, OB-C15,and OB-C16 are three pluripotent stem cells induced by Oct4 and Jhdm1band have normal karyotypes.

Refer to FIG. 4. As shown in FIG. 4, a and d are the microphotographs ofthe induced pluripotent stem cells finally formed by Oct4+Jhdm1b(abbreviated as OB); b and e are the photographs of chimeric offspringthat are developed after the induced pluripotent stem cells finallyformed by OB are injected into the blastula; c and f are photographs ofthe offspring that are generated after the chimeras mated with wild typemice, wherein the chimeras are developed after the induced pluripotentstem cells finally formed by using OB are injected into the blastula.This shows that the stem cells obtained by the method of the presentinvention can form chimera, wherein the donor cells are inducedpluripotent stem cells originated from OG2/129 cells, while thepseudopregnant mice are ICR mice fed in the laboratory. The chimeraswere capable of transmitting the original donor cells to the offspringthrough the germ line, indicating that such stem cells have goodquality.

Determination of the Functionalities of Jhdm1b Variants:

Refer to FIG. 12. As shown in FIG. 12, the Jhdm1b variants that aremutated did not have the activity of improving the reprogrammingefficiency, so the DNA binding domain (CXXC) and the catalytic domain(Jmic) of Jhdm1b are necessary for reprogramming and a lack of either ofthem will fail to promote the process of reprogramming. Furthermore, thecombination of Oct4 with Jhdm1b can complete reprogramming in a commonculture medium, and achieves more significant effects in the presence ofvitamin C.

What are described above are the embodiments of the present inventionand are not to limit the patent scope of the present invention thereto.All equivalent structures or equivalent process changes made byutilizing the description and the appended drawings of the presentinvention, or the direct or indirect applications thereof in otherrelevant technical fields, are within the patent scope of the presentinvention in the similar way.

What is claimed is:
 1. A method for generating induced pluripotent stemcell clones, the method comprising the steps of: a. introducing aretroviral expression vector consisting of (i) Oct4 and Jhdm1b or (ii)Oct4, Klf4 and Jhdm1b into mouse embryonic fibroblast; and b. culturingthe fibroblasts in an inducing medium comprising vitamin C to inducepluripotent stem cell clones.
 2. The method of claim 1, wherein theretroviral expression vector is a pMXs vector.
 3. A method forgenerating induced pluripotent stem cell clones, the method comprisingthe steps of: a. introducing a retroviral expression vector consistingof (i) Oct4, Jhdm1a and Jhdm1b or (ii) Oct4, Klf4, Jhdm1a and Jhdm1binto mouse embryonic fibroblasts; and b. culturing the fibroblasts in aninducing medium comprising vitamin C to induce pluripotent stem cellclones.
 4. The method of claim 1, further comprising a step c: culturingand expanding the induced pluripotent stem cell clones in a stem cellculture medium in the presence of feeder cells.
 5. The method of claim3, further comprising a step c: culturing and expanding the inducedpluripotent stem cell clones in a stem cell culture medium in thepresence of feeder cells.
 6. The method of claim 3, wherein theretroviral expression vector is a pMXs vector.
 7. The method of claim 1,wherein the inducing medium includes DMEM high glucose medium, 15% fetalbovine serum, 0.1 mM nonessential amino acid, 2 mML-glutamine, 1 mMsodium pyruvate, 55 μM β-mercaptoethanol, 50 U/mL penicillin, 50 μg/mLstreptomycin, 1000 U/ml leukemia inhibitory factor, and 50 μg/mL vitaminC.
 8. The method of claim 3, wherein the inducing medium includes DMEMhigh glucose medium, 15% fetal bovine serum, 0.1 mM nonessential aminoacid, 2 mML-glutamine, 1 mM sodium pyruvate, 55 μM β-mercaptoethanol, 50U/mL penicillin, 50 μg/mL streptomycin, 1000 U/ml leukemia inhibitoryfactor, and 50 μg/mL vitamin C.